We are highly concerned in recent years about deteriorating global environment. Technological developments for recovery and protection of environment are urgently needed. There exists a consensus that environmental pollution is caused by progressively expanding human industrial activities, for example, waste water from factories. Meanwhile, we are now realizing that plastic products, so commonly found in our daily life that they have become indispensable in modern life, are no less impacting on environment. Many general-purpose plastics and synthetic polymers are produced chemically from petroleum. These chemical products are amazingly convenient due to their stability, light weight, strength, and low cost. Another fact about plastics is that they have been consumed and discarded without much thought for a long time. Today, many waste plastics are alleged to be threatening ecosystems because they do not decompose in natural environment. Depending how they are processed for disposal, they can be a source for dioxin and like environmental hormones (endocrine disrupting chemicals). We should not belittle the danger of plastics.
Growing awareness of environmental issues has brought biodegradability under a new spotlight, leading to the advent of a new concept “biodegradable plastics.” A quick implementation of the concept is being awaited. A promising candidate for raw material for biodegradable plastics and hydrogels is biopolymer produced by microorganisms. Especially, the potential found in a class of biopolymer called polyamino acids which consist of a chain of amino acids with special forms of linkages is attracting a lot of interest. Three polyamino acids have been identified: poly-γ-glutamate (“PGA”), poly-ε-lysine, and cyanophycin.
Recent studies have revealed that the structural properties of polyamino acid (optical activity, type, molecular size, forms of linkages, etc. of constituent amino acid) strongly affect the functionality of the polyamino acid. PGA is a polyamino acid formed by amide bonding between α-amino groups and γ-carboxyl groups of glutamate. PGA is now well-known as the main substance of the sticky threads of Natto. The stickiness is largely due to the functionality in question. PGA is well-known for having both biodegradability and high water absorption. These functions are expected to find various applications in the food, cosmetics, medical products, and many other fields. A drawback of the currently commercialized PGA is that they are produced from Bacillus subtilis var. natto or their analogues. The result is chemically heteropolymers with both the optical isomers of the glutamate linked in a random manner. This fact presents a large obstacle in evaluating practical use of PGA as an alternative raw material to plastics.
There is a report about homopoly-γ-glutamate producing bacteria. For example, Non-patent Document 1 reports that Bacillus anthracis produces a poly-γ-D-glutamate consisting only of D-glutamate (“D-PGA”). However, the bacterium is highly pathogenic, hence unsuitable for use in PGA production on an industrial scale. Furthermore, the D-PGA produced has a low molecular weight. There is another report (Non-patent Document 2) that an alkalophilic bacterium, Bacillus halodurans, produces a poly-γ-L-glutamate (“L-PGA”) consisting only of L-glutamate. However, the L-PGA produced by the bacterium also has a very low molecular weight.
A homopoly-γ-glutamate having relatively large molecular weight is reported to be produced by a halophilic archaebacterium, Natrialba aegyptiaca, which produces only poly-γ-L-glutamate having a molecular weight approximately from 100,000 to 1,000,000. This bacterium, however, has a molecular weight as low as about 100,000 in liquid culture. Also, the bacterium produces little poly-γ-L-glutamate and unsuited for industrial use. See Non-patent Document 3 and Patent Document 1.
Another poly-γ-L-glutamate producing organism is the hydra. The hydra however has the same, very low molecular weight problem. See Non-patent Document 4.
An application field for PGA is cosmetics. In applying PGA to cosmetics, PGA (water-soluble polymer compounds, in general) is required to have properties, such as uniform optical purity as well as high moisture retention and viscosity enhancement. To satisfy these two requirements at the same time, it is desirable that PGA should have uniform optical purity and large molecular weight.
Water absorbent resin is used in numerous fields: e.g., in disposable diaper and sanitary goods, for medical, construction, civil engineering, and architectural purposes, as texture enhancer, freshness-keeping agents for food, and important base materials for green engineering in the agricultural field such as gardening.
Among water absorbent resins, the acrylic ones are used in various fields owing to their excellent water absorption and low price. However, the acrylic water absorbent resins are hardly biodegradability. It is therefore difficult to process the acrylic water absorbent resins through decomposition by microorganisms. For example, they are not suitable for compost production or similar biological processing. When used in land filling, they remain there without decomposing.
Water absorbent resins addressing these problems are suggested. Patent Document 1, for example, discloses a biodegradable water absorbent resin composed of crosslinked poly-γ-glutamate. PGA is a polymer compound synthesized by various organisms and highly biodegradable. Patent Document 1 therefore evaluates the biodegradable water absorbent resin as being safely and easily disposable.
To summarize the discussion about conventional PGAs, most of them are formed from irregular linking of the two optical isomers, L-glutamate and D-glutamate, as is the case with the PGA in Patent Document 2. Some of the reported PGAs are formed from linking of only D-glutamate (Non-patent Document 1) and of only L-glutamate (Patent Document 1, Non-patent Documents 2 to 4).
In this specification, for convenience of description, PGA formed by the linking between D-glutamate and L-glutamate will be referred to as DL-PGA, PGA formed only from D-glutamate as D-PGA, and PGA formed only from L-glutamate as poly-γ-L-glutamate or L-PGA.
The biodegradable water absorbent resin of Patent Document 2 has a problem that the biodegradable water absorbent resin is difficult to stably produce with desired quality. It is also difficult in the first place to produce the crosslinked DL-PGA which constitutes the biodegradable water absorbent resin.
More specifically, the DL-PGA, or the starting material for the crosslinked DL-PGA disclosed in Patent Document 2, is synthesized by a Natto bacterium (e.g., Bacillus subtilis) or its analogue. In the DL-PGA obtained from a Natto bacterium or its analogue, however, D-glutamate and L-glutamate form irregular linkages; the content ratio and sequence of the D-glutamate and the L-glutamate change every time the PGA-producing bacterium is cultured. The crosslinked DL-PGA therefore has a different structure, hence different properties, from one molecule to the other. That will likely lead to quality difference depending on lots of the DL-PGA used in the production of the crosslinked substance, making it difficult to stably produce crosslinked PGA with desired quality.
Furthermore, it is generally believed that the starting material, DL-PGA, with inconsistent quality as in the case above makes it difficult to stably produce a crosslinked substance. The inventors of the present invention could not obtain crosslinked DL-PGA in research. This is presumably because DL-PGA, as mentioned earlier, has a different structure from one molecule to the other. In other words, the crosslinking efficiency in the production of crosslinked PGA depends on molecular structure. If individual molecules have an irregularly different structure, the crosslinking efficiency drops markedly. It is therefore difficult to crosslink DL-PGA in which each molecule has a different structure, and the yield of the crosslinked substance is very low.
Up until now, there are no reports at all that crosslinked L-PGA has been successfully obtained. This is presumably for the following reasons. No conventional liquid culture has successfully produced L-PGA with large average molecular weight. It is also a common technical knowledge that it is extremely difficult to obtain a crosslinked organic compound with a low molecular weight. These facts are so prohibitive that the person skilled in the art would not even conceive of obtaining low molecular weight crosslinked L-PGA. The result is a total lack of reports of attempts to obtain crosslinked L-PGA. Industrial purpose PGA is required to be producible by liquid culture because plate culture is hardly capable of producing large amounts of microorganisms, and collecting L-PGA from plate culture media is not efficient.
As an exemplary L-PGA synthesizing organism, Non-patent Document 1 discloses an alkalophilic bacterium, Bacillus halodurans, and Non-patent Document 2 discloses hydra. These organisms however can only synthesize L-PGA with very low molecular weights (no greater than 100,000).
Patent Document 2 and Non-patent Document 3 report that Natrialba aegyptiaca, a halophilic archaebacterium, produces L-PGA with molecular weights of about 100,000 to 1,000,000 if cultured on plate culture media. The L-PGA synthesized by Natrialba aegyptiaca in liquid culture, however, has a molecular weight of about 100,000, and its synthesis efficiency is very low.
Crosslinked D-PGA, even if ever obtained, is not suitable for industrial use.
A major reason is that the D-PGA synthesizing bacterium disclosed in Non-patent Document 4 is highly pathogenic Bacillus anthracis. The use of Bacillus anthracis in PGA producing for industrial purposes is utterly unsuitable.
There are two causes for rough skin. One is the peeling off of keratin cells. The other is deteriorating conditions of the skin in a dry atmosphere, which could lead to hardening of, hence damage to, epidermis. The rough skin due to desquamated keratin cells is caused, for example, by elution of intercorneocyte lipid, such as cholesterol, ceramide, and fatty acid; denaturation of keratin cells by ultraviolet rays and detergent; and hypoplasia of a keratin layer transmission barrier caused by interruption of balanced growth of epidermic cells and/or balanced keratinization.
Research activities have been underway about the synthesis of lipid components between keratin cells or similar intercorneocyte lipid and the delivery of the lipid components to the skin, for the purpose of prevention or treatment of rough skin. Lamella granules are biosynthesized by cells in a prickle layer and a granular layer and released between cells beneath a keratin layer, spreading to form a lamella structure. This substance present between cells is called intercorneocyte lipid.
Lamella granules contain, among other substances, glucosylceramide, cholesterol, ceramide, and phospholipid. Intercorneocyte lipid contains little glucosylceramide. In other words, the glucosylceramide in lamella granules is thought to be hydrolyzed by β-glucocerebrosidase and converted to ceramide. The ceramide then forms a lamella structure to facilitate the formation of keratin transmission barrier as an intercorneocyte lipid, acting as a barrier preventing rough skin. Especially, ceramide supplementation is reported in Non-patent Document 5 to be highly effective to rough skin caused by detergent and like material.
Meanwhile, to prevent rough skin due to hardened or damaged epidermis, external dermal agents with moisture retention effect, such as cosmetics, have been conventionally used. Use of an external dermal agent with moisture retention effect prevents evaporation of water via skin, allowing the epidermis and keratin layer to retain water. The function preserves the skin's homeostasis, hence moisture retention capability and softness, keeping the skin young and fresh.
Examples of conventionally reported lipophilic substances with skin moisture retention effect include vegetable oils, such as olive oil, and animal lipids, such as lanolin. Examples of hydrophilic substances with skin moisture retention effect include water-soluble polyhydric alcohols, such as glycerine, 1,3-butylene glycol, propylene glycol, and sorbitol; polysaccharides, such as hyaluronic acid and xanthan gum; water-soluble polymers, such as polyethylene glycol; salt of pyrrolidone carboxylic acid; natural moisture retention factors with low molecular weight (amino acid is a typical example); and vegetable extracts.
Like above examples, there are numerous kinds of substances with skin moisture retention effect. Those derived from animals and chemically synthesized are however avoided in recent years to follow the social trend for improved safety. For the same reason, substances derived from natural products and those obtained by fermentation by microorganisms are considered better. Furthermore, biodegradable materials, having much less negative impact not only on living things but also on environment, are regarded as being promising and receiving much attention.
Among biodegradable materials, the biopolymer produced by microorganisms is viewed as having good prospects. Especially, it has been discovered that a class of biopolymers called polyamino acid formed by condensation polymerization of amino acid have various functions and are receiving much attention for their potential capabilities. PGA, one of the polyamino acids, is of especially high interest.
PGA is a polyamino acid formed by amide bonding between α-amino groups and γ-carboxyl groups of glutamate, as mentioned earlier. PGA is a water absorbent polyamino acid known as the main substance of the sticky threads of Natto, a traditional Japanese favorite. The Japanese have a liking for Natto largely because of its attractive functionality. A known attractive function of PGA is a combination of biodegradability and high water absorption. Exploiting these functions, PGA is expected to find applications not only as cosmetics material as mentioned above, but also in the medical, food, and various other fields.
Nevertheless, some issues persist with the external dermal agents containing the conventional PGA. These agents are difficult to stably produce with desired quality and provide insufficient moisture retention.
The DL-PGA currently available as commercial products is chemically heteropolymers as mentioned earlier. Specifically, PGA is produced from a Natto bacterium or its analog. D-glutamate and L-glutamate form irregular linkages. The content ratio and sequence of the glutamates change every time the PGA-producing bacterium is cultured. Generally, the structural properties of polyamino acid (optical activity, type, molecular size, forms of linkages, etc. of constituent amino acid) strongly affect the functionality of the polyamino acid. The DL-PGA has a different structure, hence different properties, from one molecule to the other. That makes it difficult to stably produce DL-PGA with desired quality.
Furthermore, the DL-PGA, having insufficient moisture retention capability, poses large problems in developing commercial external dermal agents (e.g., cosmetics).
Up until now, there are no reports at all that an L-PGA-containing external dermal agent has been successfully produced. This is presumably for the following reasons.
Generally, when an external dermal agent is produced containing PGA, the PGA must have large molecular weight because the PGA is required to provide moisture retention capability. On the other hand, no conventional liquid culture has successfully produced L-PGA with large average molecular weight. This fact is so prohibitive that the person skilled in the art could not even conceive of producing an L-PGA-containing moisture retention agent.
In addition, as mentioned earlier, industrial purpose PGA is required to be producible by liquid culture. It is difficult to culture large amounts of microorganisms in a single process by plate culture, and collecting L-PGA from plate culture media is not efficient. In addition, D-PGA is not suitable for industrial use as mentioned earlier.
In Patent Document 2, crosslinked DL-PGA is used as a water absorbent resin. It is however difficult to use crosslinked DL-PGA as an external dermal agent.
The DL-PGA disclosed in Patent Document 2, the starting material for the crosslinked DL-PGA, is synthesized by a Natto bacterium (e.g., Bacillus subtilis) or its analogue. This method cannot be free from the inconsistent quality of the starting material (DL-PGA) and hardly produces a crosslinked substance in a stable manner. The inventors of the present invention could not obtain crosslinked DL-PGA in research. This is presumably because DL-PGA, as mentioned earlier, has a different structure from one molecule to the other. In other words, the crosslinking efficiency in the production of crosslinked PGA depends on molecular structure. If individual molecules have an irregularly different structure, the crosslinking efficiency drops markedly. It is therefore difficult to crosslink DL-PGA in which each molecule has a different structure, and the yield of the crosslinked substance is very low.
Thus, it is difficult to stably produce an external dermal agent with desired quality even by using crosslinked DL-PGA.
Meanwhile, up until now, there are no reports at all that crosslinked L-PGA has been successfully obtained.
This is because liquid culture has been never successful in producing L-PGA with large average molecular weight as mentioned earlier. It is a common technical knowledge that it is extremely difficult to obtain a crosslinked organic compound with a low molecular weight. These facts are so prohibitive that the person skilled in the art would not even conceive of obtaining low molecular weight crosslinked L-PGA. The result is a total lack of reports of attempts to obtain crosslinked L-PGA.
Crosslinked D-PGA, if ever obtained, is not suitable for industrial use because the only currently known D-PGA producing bacterium is Bacillus anthracis as mentioned earlier.
[Patent Document 1]
Published Japanese Translation of PCT Application No. 2002-517204 (Tokuhyo 2002-517204; published Jun. 18, 2002)
[Patent Document 2]
Japanese Unexamined Patent Publication No. 10-251402/1998 (Tokukaihei 10-251402; published Sep. 22, 1998)
[Non-Patent Document 1]
Makino, S., I. Uchida, N. Terakado, C. Sasakawa, and M. Yoshikawa, Molecular characterization and protein analysis of the cap region, which is essential for encapsulation in Bacillus anthracis, Journal of Bacteriology, 1989, 171, 722-730.
[Non-Patent Document 2]
Aono, R., M. Ito, and T. Machida, Contribution of the Cell Wall Component Teichuronopeptide to pH Homeostasis and Alkaliphily in the Alkaliphile Bacillus lentus C-125, Journal of Bacteriology, 1999, Vol. 181, 6600-6606.
[Non-Patent Document 3]
Hezayen, F. F., B. H. A. Rehm, B. J. Tindall and A. Steinbuchel, Transfer of Natrialba asiatica B1T to Natrialba taiwanensis sp. nov. and description of Natrialba aegyptiaca sp. nov., a novel extremely halophilic, aerobic, non-pigmented member of the Archaea from Egypt that produces extracellular poly(glutamic acid), International Journal of Systematic and Evolutionary Microbiology, 2001, 51, 1133-1142.
[Non-Patent Document 4]
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[Non-Patent Document 5]
Skin and Beauty, 36, 210 (2004)